Apparatus and process for fermentation of biomass hydrolysate

ABSTRACT

A process for converting biomass hydrolysate into biofuel, the process comprising the steps of: obtaining a biomass hydrolysate solution comprising monosaccharides; immobilizing  Pachysolen tannophilus ; contacting the solution with the immobilized  Pachysolen tannophilus ; and recovering a fermented biofuel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.13/869,842, filed Apr. 24, 2013 which is a Continuation of U.S.application Ser. No. 12/856,566, filed Aug. 13, 2010 which claims thebenefit of U.S. Provisional Application No. 61/233,821, filed Aug. 13,2009, which are hereby incorporated by reference.

FIELD

The present patent document relates to an apparatus and process forfermentation of biomass hydrolysate.

BACKGROUND

Recently, conversion of biomass through saccharification andfermentation into ethanol or other useful products as a replacement forfossil fuels has garnered considerable attention. Because biomass is arenewable resource typically rich in polymers of hexoses and pentoses,it is a promising substrate for fermentation.

Biomass for such conversion processes may be potentially obtained fromnumerous different sources, including, for example: wood, paper,agricultural residues, food waste, herbaceous crops, and municipal andindustrial solid wastes to name a few.

Biomass is made up primarily of cellulose and hemicellulose bound upwith lignin. The lignin inhibits the conversion of the biomass intoethanol or other biofuels, and, as a result, typically a pretreatmentstep is required to expose the polysaccharides, cellulose andhemicellulose. Once hemicellulose and cellulose are exposed,saccharification, either enzymatic or chemical, may be performed tobreak the polysaccharides into their constituent monosaccharidemonomers. Pretreatment and saccharification are used, therefore, tobreak down the long polysaccharide chains and free the sugars beforethey are fermented for biofuel production. Fermentation can begin oncefree sugars are present, either because they are naturally present orbecause a portion of the biomass has been reduced to its componentsugars, or both.

In order to be effective, current pretreatment and saccharificationprocesses attempt to liberate the biomass sugars while also minimizingthe formation of secondary products from the degradation ofhemicellulose, cellulose, and lignin, because of the inhibitory effectssecondary products may have on the subsequent fermentation processes.The presence of inhibitory secondary products has historicallycomplicated ethanol production and increased the cost of production dueto elaborate detoxification steps.

Although numerous techniques for pretreatment and saccharificationexist, the most popular methods, and the most cost effective methods,including acid hydrolysis, produce secondary products in addition tosugars, that are inhibitory to fermentation. Inhibitory secondaryproducts created as a result of the degradation of hemicellulosepentoses and hexoses include furfural and 5-hydroxymethylfurfural (HMF),respectively. Furfural and HMF may further be broken down intolevulinic, acetic, and formic acids. Other inhibitory secondary productsinclude phenolic compounds produced from the degradation of lignin andacetic acid produced by cleavage of acetyl groups within thehemicellulose. Concentrations of inhibitory secondary products in thehydrolysate will vary based on the source of the biomass and thehydrolysis method used.

Some of the secondary products formed from the breakdown ofhemicellulose, cellulose and lignin are in themselves valuablesubstances. The inventors have realized that recovery of high-valuesecondary products from the hydrolysate can improve the economics of thebiomass to biofuel process.

Other secondary products are not formed from chemical decomposition, butmay be extracted from the biomass during pretreatment and hydrolysis.These extracted secondary products include terpenes, sterols, fattyacids, and resin acids. These extracted compounds may also be inhibitoryto fermentation.

Inhibitory secondary products may be detrimental to the fermentationprocess, particularly as their concentration increases. Thus, it wouldbe advantageous if a process could be developed that allows specificmicrobes, like yeast for example, to efficiently convert biomasshydrolysate into biofuels, such as ethanol, in the presence ofinhibitory secondary products formed during pretreatment and hydrolysis.

Many inhibitory products have compound impacts when present with otherinhibitory compounds; thus, a non-inhibitory amount of a certaincompound may become inhibitory in the presence of a second inhibitorycompound. Furthermore, even following partial recovery and/or removal ofinhibitory secondary products, the remaining concentrations may beinhibitory to fermentation due to these synergies. Thus, it would beadvantageous if a process could be developed that allows specificmicrobes, like yeast for example, to efficiently convert biomasshydrolysate into biofuels, such as ethanol, in the presence ofinhibitory secondary products formed during pretreatment and hydrolysis,even when the concentrations of the individual inhibitory secondaryproducts are below their respective inhibitory concentration level buttheir combined concentration is inhibitory.

Cellulose is a homogeneous polysaccharide composed of linearly linkedglucose units. Glucose is a hexose, which may be readily fermented by anumber of microbes including Saccharomyces cerevisiae (traditionalbaker's yeast) and Kluyveromyces marxianus. Yeast cells are especiallyattractive for cellulosic ethanol processes, as they have been used inbiotechnology for hundreds of years, are tolerant to high ethanol andinhibitor concentrations, and can grow at low pH values. A low pH valuehelps avoid bacterial contamination and is therefore advantageous.

Unlike cellulose, hemicellulose is a heterogeneous polymer of pentoses,hexoses, and uronic acids. The saccharides principally found inhemicellulose are the pentoses xylose and arabinose and the hexosesglucose, mannose and galactose. The relative amounts of differentpentoses and hexoses vary with the biomass type. The hemicellulosecontent of some cellulosic biomass may reach as high as 38% or more ofthe total dry biomass weight. Therefore, hemicelluloses, and thepentoses and hexoses they contain, may comprise a substantial portion ofthe convertible sugars available in the biomass. As a result, in orderto improve the economics of the biomass to biofuel conversion process,much research has been performed on identifying microorganisms thatefficiently convert pentoses and hexoses to biofuel, such as ethanol.

While numerous microbes have been found to process hexoses into ethanol,efficiently fermenting pentoses has proven more elusive. Some bacteriaand fungi can inefficiently convert pentoses to ethanol and manymicrobes can only process pentoses when assisted by enzymes. For a longtime it was thought that yeast strains could not anaerobically fermentpentoses. However, U.S. Pat. No. 4,359,534 to Kurtzman et al. disclosesthe use of Pachysolen tannophilus to ferment pentoses. Similarly, U.S.Pat. No. 7,344,876 to Levine discloses a pure culture of Kluyveromycesmarxianus capable of proliferation on pentoses as the sole carbonsource.

While the patents to Kurtzman and Levine disclose the use of yeasts forfermentation of pentoses into ethanol, commercial applications have beenlimited because of the detrimental effects of inhibitory secondaryproducts typically found in biomass hydrolysate. Yeasts that can fermentxylose and other pentoses in an artificial, or controlled, mediumgenerally perform poorly in acid hydrolysates. Challenges presented bybiomass hydrolysate include an acidic pH and a high concentration oftoxic compounds, including acetic acid, phenolic compounds,5-hydroxymethylfurfural (HMF) and furfural, and other inhibitorymolecules produced during hemicellulose hydrolysis.

Because of the detrimental effects of inhibitory secondary products onthe production of ethanol, biomass hydrolysate is currently subjected toa conditioning process after pretreatment and hydrolysis to reduce theconcentration of inhibitory secondary products. This conditioningprocess adds complexity and cost to the overall process and reduces theefficiency and cost-effectiveness of the conversion process.Furthermore, the greater the required reduction in the concentrationlevels of the inhibitory secondary products, the greater the complexityand cost. A need, therefore, exists for a process in which microbes,such as different yeast strains, could more effectively convertpentoses, as well as hexoses, into ethanol and other biofuels in thepresence of inhibitors formed during the pretreatment and hydrolysisprocess. In addition, it would be beneficial to develop schemes wherebyinhibitory secondary products may be partially recovered and purified,instead of only removed and discarded, from hydrolysate.

Furthermore, if an efficient method for converting pentoses to ethanolexisted, the discarded hemicellulose in the paper pulping process mightbe converted into alcohol instead. Similarly, sugar cane residues,referred to as bagasse, could also be subjected to hemicelluloseconversion prior to being combusted for their fuel values. Thepossibility of removing hemicellulose from the paper pulping process andconverting it to ethanol was hypothesized by the Georgia Institute ofTechnology in W. J. Fredrick et al., Co-production of ethanol andcellulose fiber from Southern Pine: A technical and economic assessment,32 Biomass and Bioenergy 1293-1302 (2008). However, the GeorgiaInstitute of Technology process explicitly requires the hydrolysate tobe conditioned to remove inhibitors and noted the lack of an efficientprocess to convert pentoses into ethanol. The study noted that“Fermentation is carried out after inhibiting contaminants have beenremoved from the hydrolysate.” The study further notes that the 85%conversion factor of pentoses to ethanol “is an optimistic estimate thatassumes that on-going research will make it possible . . . ” The studyconcludes that ethanol production from loblolly pine may not becompetitive with ethanol from other lignocellulosic sources when it isco-produced with cellulose fiber.

SUMMARY OF THE INVENTION

In view of the foregoing, an object according to one aspect of thepresent patent document is to provide an improved apparatus and processfor converting biomass hydrolysate into ethanol or other biofuel.Preferably the apparatus and process address, or at least ameliorate oneor more of the problems described above. To this end, a process forconverting biomass hydrolysate into biofuel is provided; the processcomprises the steps of: obtaining a biomass hydrolysate solutioncomprising monosaccharides; immobilizing a fermentative microbecontacting the solution with the immobilized fermentative microbe; andrecovering a fermented biofuel. The recovered biofuel preferablycomprises alcohol, and more preferably comprises ethanol.

In another embodiment, a process for converting biomass hydrolysate intobiofuel is provided comprising the steps of: contacting a biomasshydrolysate solution with immobilized fermentative microbe strain for asufficient reaction time to convert monosaccharides in the biomasshydrolysate to biofuel; and recovering biofuel from the fermentedhydrolysate.

In certain implementations of the foregoing embodiments, thefermentative microbe is Pachysolen tannophilus and Pachysolentannophilus is immobilized in calcium alginate. The calcium alginate maybe in the form of beads ranging from 0.1 mm to 5 mm in diameter, and aremore preferably about 2 mm to 3 mm in diameter. The calcium alginate isnot required to be in bead form and may be in any other form thatpermits the Pachysolen tannophilus to be immobilized but still allowsthe sugar substrates in the biomass hydrolysate to kinetically interactwith the yeast. For example, the calcium alginate may be in a sponge ormesh form. Similarly, the Pachysolen tannophilus/calcium alginatemixture may be applied as a coating to a natural or synthetic matrix toincrease the surface area per mass of Pachysolen tannophilus/calciumalginate mixture.

Preferably, the immobilized culture of Pachysolen tannophilus isperiodically treated with a yeast growth medium to restore metabolicefficiency to the Pachysolen tannophilus. The metabolic efficiency maybe lost over long periods of use, especially in connection withcontinuous flow bioreactors.

In another embodiment, the immobilized fermentative microbe strain is atleast one microbe selected from a group consisting of Pichia, Candida,Klyveromyces and Zymomonas mobilis NREL strain 8b.

In yet another embodiment, the alginate used to immobilize the cultureof Pachysolen tannophilus is periodically recovered and recycled bytreating the Pachysolen tannophilus/calcium alginate with a calciumchelator and monovalent counter-ion, such as sodium citrate. Theresulting dialysis of the solution with an inorganic salt, such assodium chloride, regenerates sodium alginate, from which calciumalginate may be regenerated.

In yet another embodiment, the biomass hydrolysate contains asubstantial amount of secondary products that inhibit fermentation. Thehydrolysate solution may contain furfural levels in the range of about0.01 to 10 g/L, 5-hydroxymethylfurfural levels in the range of about0.01 to 10 g/L, and acetic acid levels in the range of about 0.05 to 20g/L, or even 0.5 to 20 g/L. In addition, the hydrolysate solution maycontain phenolic compounds in the range of about 0.01 to 10 g/L. Theselevels of furfural, HMF, phenolic compounds, and acetic acid may occurin combination or in isolation. Other inhibitors may also be present.

In yet another embodiment, more than 80% of the monosaccharides in thesolution are converted to ethanol.

In still another embodiment, the biomass hydrolysate is obtained fromthe biomass by pressing. The biomass and biomass hydrolysate may besubjected to a high pressure press capable of squeezing thesugar-containing liquid forming the biomass hydrolysate out of thebiomass residue.

In other embodiments, the biomass hydrolysate may be conditioned bypassing the hydrolysate over activated carbon, a strong acid ionexchange resin and/or a weak base ion exchange resin.

In the various embodiments described above, the biomass hydrolysatesolution may contains inhibitory secondary products sufficient toprevent more than 50% conversion of pentoses by the fermentativemicrobes in their “free” state.

In another aspect, a process for converting biomass hydrolysate intobiofuel is provided comprising the steps of: contacting the biomasshydrolysate solution with a first immobilized microbe strain; contactingthe biomass hydrolysate solution with a second immobilized microbestrain; and recovering a fermented biofuel.

In one embodiment the first immobilized microbe strain is a bacteriumand the second immobilized microbe strain is a yeast. Further, the firstimmobilized microbe strain may be contained in a first reactor and thesecond immobilized microbe strain may be contained in a second reactor.In an alternative embodiment, both immobilized microbe strains may be inthe same reactor. If implemented so both strains are in the samereactor, the first immobilized microbe strain and the second immobilizedmicrobe strain may also be immobilized together within the sameimmobilization medium.

Preferably, the immobilization medium is a calcium alginate bead, butother immobilization mediums may also be used. Further, the firstimmobilized microbe strain may be immobilized in a first immobilizationmedium and the second immobilized microbe strain may be immobilized in asecond immobilization medium.

In one embodiment, the second immobilized microbe strain is capable offermenting mannose to a biofuel.

In yet another aspect of the present patent document, a process forconverting biomass hydrolysate into biofuel is provide comprising thesteps of: flowing a biomass hydrolysate solution comprisingmonosaccharides and one or more inhibitory secondary products through acontinuous flow reactor containing an immobilized microbe strain andcontacting the immobilized microbe strain with the biomass hydrolysate;and recovering a fermented biofuel.

In one embodiment, the flow rate of the biomass hydrolysate is set toexceed the sedimentation rate of the immobilized microbe strain in a“free” condition. Preferably, the continuous flow reactor is an upflowreactor, but other continuous reactors may also be used.

In another embodiment, the productivity of the biofuel conversionprocess is at least 0.3 g/L·h for a flow rate corresponding to a 10 hourretention time. In still another embodiment, the productivity of thebiofuel conversion process is at least 0.42 g/L·h for a flow ratecorresponding to a 5 hour retention time.

In a further aspect, a medium for fermenting biomass hydrolysate isprovided. In one embodiment, the medium comprises calcium alginate beadsranging from 0.1 mm to 5 mm in diameter, and a microbe strain capable offermenting pentoses immobilized in the calcium alginate beads, whereinthe immobilized microbe strain is capable of converting at least 70% ofavailable pentoses in a biomass hydrolysate to a biofuel.

In yet another aspect, a medium for fermenting biomass hydrolysate isprovided, comprising an immobilization substance capable of providing amicro environment for a microbe strain; and a microbe strain capable offermenting pentoses into a biofuel immobilized in the immobilizationsubstance, wherein the microbe strain comprises about 5% by volume ofthe immobilization substance.

As described more fully below, the apparatus and processes of thepresent patent document permit the efficient conversion of biomasshydrolysate into ethanol, even in the presence of high levels ofinhibitory secondary products formed or extracted during pretreatmentand/or fermentation steps of the process. Further aspects, objects,desirable features, and advantages of the methods disclosed herein willbe better understood from the detailed description and drawings thatfollow in which various embodiments are illustrated by way of example.It is to be expressly understood, however, that the drawings are for thepurpose of illustration only and are not intended as a definition of thelimits of the claimed invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an overview of one embodiment of a process for theconversion of biomass into a biofuel such as ethanol.

FIG. 2 illustrates an overview of another embodiment of a process forthe conversion of biomass into biofuels such as ethanol.

FIG. 3 illustrates a process for recycling a calcium alginateimmobilization medium.

FIG. 4 illustrates a view of one embodiment of a bioreactor forperforming submerged fermentation of biomass hydrolysate usingimmobilized microbes.

FIG. 5A illustrates a side view of another embodiment of a bioreactorfor performing submerged fermentation of biomass hydrolysate usingimmobilized microbes.

FIG. 5B illustrates a front view of the bioreactor shown in FIG. 5A.

FIG. 6 illustrates an up-flow reactor for performing submergedfermentation of biomass hydrolysate using immobilized microbes.

FIG. 7 is a graph illustrating ethanol yield of regenerated calciumalginate beads with immobilized fermentative microbes over a series offermentations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Consistent with its ordinary meaning as a renewable energy source, theterm “biomass” is used herein to refer to living and recently deadbiological material including carbohydrates, proteins and/or lipids thatmay be converted to fuel for industrial production. By way ofnon-limiting example, “biomass” refers to plant matter, including, butnot limited to switchgrass, sugarcane bagasse, corn stover, corn cobs,alfalfa, Miscanthus, poplar, and aspen, biodegradable solid waste suchas dead trees and branches, yard clippings, recycled paper, recycledcardboard, and wood chips, plant or animal matter, and otherbiodegradable wastes.

The present patent document teaches new and improved processes andapparatuses for fermenting biomass hydrolysate. Processes used toconvert polysaccharides in biomass into hexoses and pentoses oftencreate inhibitory secondary products that prevent or hinderfermentation. Furthermore, the combinations of inhibitory secondaryproducts found in actual biomass hydrolysate are more toxic to fermentsthan any single inhibitory secondary product added to a defined,artificial medium. The present patent document teaches novel processesthat increase the tolerance of the fermentative microbes to inhibitorysecondary products found in biomass hydrolysate by immobilizing themicrobes. In certain embodiments, fermentation of hemicellulosehydrolysate containing inhibitory secondary products is carried outusing immobilized Pachysolen tannophilus. In some embodiments,fermentation of hemicellulose hydrolysate is carried out using animmobilized microbe, even though the concentration of an individualsecondary product or the combined concentration of secondary products inthe biomass hydrolysate would be inhibitory to the microbe in its freestate.

Immobilization confers an increased resistance on microbes to inhibitorysecondary products. For example, immobilization in a calcium alginategreatly reduces the susceptibility of the yeast Pachysolen tannophilusto inhibitors contained in softwood hydrolysate. The benefits ofimmobilization, however, are not limited to Pachysolen tannophilus.Indeed, numerous different microbes may benefit from immobilizationincluding, for example, yeasts from the genera Pichia, Candida, andKlyveromyces. In addition, bacterium microbes such as Zymomonas mobilis,NREL strain 8b, also show an increased resistance to inhibitorysecondary products when immobilized.

Preferably the calcium alginate, or other material used to immobilizethe microbes, is in a form with a high surface area such as in bead,sponge, or mesh form. In addition, the immobilized microbe orcombination of microbes should also be able to ferment monosaccharidesfound in hemicellulose hydrolysates—including the hexoses mannose,galactose and glucose and the pentoses xylose and arabinose—to biofuelwith high efficiency.

FIG. 1 illustrates a general overview of one embodiment of a process forconverting biomass to ethanol or other biofuels. The primary stepsinclude pretreatment 100, hydrolysis 102, fermentation 104, and biofuelrecovery 106. FIG. 2 illustrates another embodiment of a process forconverting biomass to ethanol or other biofuels. The process in FIG. 2differs from that in FIG. 1 in that it also includes a solid/liquidseparation step 108, an optional evaporation step 112, an optionalconditioning step 110, and an optional secondary product recovery step114. If the biomass hydrolysate is provided from another source insteadof generated on site, the process of the present patent document may becondensed to performing step 104 or steps 104 in combination with step106.

Before biomass can be fermented, it often needs to undergo some form ofprocess to disrupt the polymer network of cellulose, hemicellulose, andlignin forming the biomass structure so the polysaccharides can bereduced to monosaccharides. This process is commonly referred to as“pretreatment” and is designed to reduce the recalcitrance of thebiomass to enzymatic or chemical saccharification of the cellulose andhemicellulose, therein. The pretreatment step 100 may occur through anumber of methods, including for example, in a pressure reactor. Table 1lists appropriate ranges for temperature, dwell time, and moisturecontent suitable for pretreatment in a pressure reactor. However, otheroperating conditions may also be suitable.

TABLE 1 Pressure Reactor Pretreatment Conditions Temperature* 105-200°C. Time 1 minute-24 hours Moisture Content 25-95% *Temperature dictatesthe pressure in a sealed vessel assuming a saturated steam system

Effectiveness of the pretreatment step 100 may be increased by addingone or more reagents. Reagents may include, but are not limited to:nitric acid, phosphoric acid, hydrochloric acid, sulphuric acid, sulphurdioxide, and sodium sulphite. Other reagents that reduce therecalcitrance of the biomass to hemicellulose removal may also be added.

In addition to performing pretreatment 100 in a pressure reactor,pretreatment 100 may be performed using a number of other methods,including acid prehydrolysis, steam cooking, alkaline processing,rotating augers, steam explosion, ball milling, or any other method thatreduces the recalcitrance of the biomass to saccharification of thecellulose and hemicellulose contained therein.

Once the cellulose and hemicellulose are exposed through pretreatment100, the polysaccharides are broken down into their monosaccharidecomponents so they can be fermented.

The Hydrolysis step 102 is used for converting the polysaccharides intofermentable sugars. In some of the harsher pretreatments 100, hydrolysis102 may occur simultaneously with the pretreatment step 100 and aseparate hydrolysis step 102 is not required. The two basic forms ofhydrolysis 102 are thermo-chemical and enzymatic. Thermo-chemicalhydrolysis is typically performed using a concentrated acid such assulfuric acid or hydrochloric acid at relatively low temperatures or byusing a dilute acid at relatively high temperatures.

Once the monosaccharides have been generated through the hydrolysis step102, fermentation can begin. Although fermentation can occur within thebiomass residue with some fermentation techniques, in the processesdescribed in the present patent document, a biomass hydrolysate solutioncomprising monosaccharides will typically be obtained by pressing and/orwashing the biomass residue. The obtained biomass hydrolysate is thenfermented ex-situ in fermentation step 104.

Recovery of the sugars from the biomass residue is preferably achievedthrough solid-liquid separation. For example, as shown in FIG. 2, asolid-liquid separation step 108 may be used to recover the sugars fromthe biomass residue. Solid-liquid separation may be performed using anumber of methods including, but not limited to, centrifuging orpressing. Preferably, pressing may be accomplished with a hydraulicpress. However, numerous types of mechanical or machine presses may beused. For example, a mechanical press such as a conventional screwpress, a hydro-mechanical press, a pneumatic press or any other type ofpress that can apply the necessary pressure to remove the hemicellulosehydrolysate from the cellulose/lignin residue may be used. The press mayhave a range of capabilities and configurations for pressing out thehemicellulose hydrolysate. Preferably the press can generate from atleast about 10.5 kg/cm² to about 21.1 kg/cm². In other embodiments, itis desirable if the press can generate at least approximately 1,410kg/cm².

Pressing has additional advantages because the biomass residue (whichwill comprise cellulose and lignin at this point) may be more valuableas a coal replacement if its density can be maximized and its moisturecontent minimized, thereby increasing its energy density. For pulp millfeed there are no requirements for moisture or density but minimizationof fiber damage is important. Pulp quality is measured based on itsfiber length, among other variables, but not moisture content. However,if a high energy density fuel replacement is made instead of paper pulp,reducing the moisture content is an important factor.

Accordingly, the final product that the biomass residue is to eventuallybe used for may determine what size and kind of press to use forsolid/liquid separation. For example, if the biomass residue is toeventually be used to generate cellulose and/or lignin fibers to makepaper products, cardboard, or fiberboard, a lower pressure, such as inthe range of 10.5 kg/cm² to 21.1 kg/cm² may be advantageous to minimizedamage to the cellulose fibers. In processes that turn the biomassresidue into high energy density fuel, higher pressures may be used tominimize the moisture content, without regard to fiber quality. As aresult, it may be desirable to employ pressures of about 1,410 kg/cm² oreven higher. In other embodiments, however, pressures within the rangeof 10.5 kg/cm² to 21.1 kg/cm² may still be used, as presses generatingthese types of pressures are readily available and comparativelyinexpensive as compared to presses that are capable generating about1410 kg/cm² of pressure. For example, presses that generate betweenabout 10.5 kg/cm² and 21.1 kg/cm² of pressure are routinely used in thewine and olive oil industries to press grapes and olives, respectively.

When sugarcane bagasse is used as the biomass from which the hydrolysateis pressed, fiber condition is generally unimportant. However, when usedas a high energy density fuel replacement, the moisture content is animportant factor. Therefore, higher, rather than lower pressures, may bedesirable for purposes of performing the solid/liquid separation step108.

Pressing is also advantageous because it reduces dilution from washwater. Using wash water to separate the hydrolysate from the biomasswill dilute the sugar stream and thus lower the resulting ethanolconcentration in the fermented hydrolysate. If wash water is used,however, dilution of the sugar stream may be mitigated by the use ofevaporators or similar machinery to reduce water content in thehydrolysate through optional evaporation step 112, shown in FIG. 2. Therecovered water from evaporation may be recycled into subsequent washprocesses. Addition of an evaporation step 112 as a process stepincreases the sugar concentration of the hydrolysate and thus theethanol concentration resulting from fermentation, which in turn reducesthe costs of distillation.

Once the monosaccharides are separated from the biomass, there are anumber of microbes that may be used for converting the monosaccharidesof the biomass hydrolysate into ethanol or other biofuels infermentation step 104. For example, if the biomass hydrolysate comprisesa cellulose hydrolysate, so as to include glucose (which is a hexose),the glucose in the hydrolysate may be fermented by a number of yeaststrains including Saccharomyces cerevisiae (traditional baker's yeast)and Kluyveromyces marxianus to name a few.

On the other hand, if the biomass hydrolysate comprises a hemicellulosehydrolysate, the hydrolysate will include the pentoses xylose andarabinose, and a lower concentration of hexoses, except in the case ofsoftwood hydrolysate. In the case of softwood hemicellulose, the hexosemannose is the major saccharide and the pentose xylose is the next mostabundant. Microbes that can convert the combination of pentoses andhexoses found in hemicellulose hydrolysate into ethanol are not asabundant as those available for cellulose hydrolysate. To convert sugarsfrom hemicellulose hydrolysate into ethanol, microbes that can convertboth five-carbon and six-carbon sugars are preferably utilized so thatall of the available constituent sugars of the hemicellulose hydrolysatemay be converted to ethanol or other biofuels. The same is true if thebiomass hydrolysate comprises a combination of cellulose hydrolysate andhemicellulose hydrolysate. Microbes that can ferment hexoses andpentoses may be derived from the genera Pachysolen, Kluyveromyces,Pichia, and Candida. Pachysolen tannophilus is preferably used infermentation of a liquid hydrolysate comprising a hemicellulosehydrolysate. In particular, when immobilized, Pachysolen tannophilus hasbeen found to effectively ferment hemicellulose hydrolysate producedfrom softwood.

In addition to immobilized yeasts, immobilized bacterium may also beused to ferment hexose and pentose sugars in biomass hydrolysate. Forexample, the recombinant bacterium Zymomonas mobilis (NREL recombinant8b) may be used to ferment hemicellulose hydrolysate produced fromsoftwood, hardwood, and/or herbaceous sources.

Microbes with complementary metabolic properties may also be combined inthe same fermentation process in step 104 to allow their complementaryproperties and abilities, such as complementary hexose and pentosefermentation capabilities or complimentary metabolic rates, to be usedtogether. For example, recombinant Zymomonas is unable to fermentmannose, the most prevalent sugar contained in softwood hydrolysate, therecombinant Zymomonas mobilis is preferably paired with a complementaryyeast or bacterium that is able to effectively ferment the hexosemannose to ethanol or another biofuel when it used to ferment softwoodhydrolysate. On the other hand, in the case of sugarcane bagasse, wherethe hydrolysate primarily comprises xylose and glucose, another microbeis not required to assist the recombinant Zymomonas to achieve asatisfactory fermentation of the contained sugars.

Other combinations of microbes are also possible including pairingdifferent bacterium together, pairing different yeasts together, pairingvarious yeasts and bacterium together, or pairing or combining anynumber of microbes with complimentary features including using anynumber of microbes at the same time. As the number of combined microbesincreases, however, their capabilities may begin to overlapsignificantly and thereby reduce the additive value of the additionalmicrobes.

Depending on the biomass and treatments employed, the pretreatment step100 and hydrolysis step 102 may yield soluble sugars from the biomass inthe form of xylose, mannose, arabinose, galactose, and glucose ready forfermentation in step 104. However, other secondary products, which areinhibitory to the fermentation step 104, are also produced or extractedfrom the biomass. The concentrations of fermentation inhibitors thatform in converting biomass to fermentable hexoses and pentoses will varydepending on the source of the biomass and the methods used for thepretreatment step 100 and the hydrolysis step 102. For example aceticacid is produced by cleavage of acetyl groups from hemicellulose. Inaddition, some of the pentoses and hexoses are degraded due todehydration into furfural and HMF. Phenolic and polyphenolic compounds(collectively “Phenolic Compounds”) are also formed from the degradationof lignin. While the generated Phenolic Compounds, furfural, HMF, andacetic acid are all potentially valuable compounds, they are alsofermentation inhibitors, and may prevent or inhibit fermentation,particularly as their concentrations increase.

In addition, Furfural and HMF degrades to produce levulinic acid, aceticacid, and formic acid, which are even more potent fermentationinhibitors. Phenolic and polyphenolic compounds produced from hydrolysisof wood hemicellulose and the concomitant lignin degradation includeguaiacol, vanillin, phenol, vanillic acid, syringic acid, salicylicacid, gentisic acid, and others. Many of these compounds, for instancevanillin and vanillic acid, are known to inhibit the growth of and/orfermentation with microbial yeasts, such as Pachysolen andSaccharomyces.

In addition to secondary products made from the degradation ofhemicellulose components, other molecules may be extracted from biomassby the pretreatment and/or saccharification conditions during thepretreatment step 100 and/or hydrolysis step 102. These extractedcompounds may include terpenes, sterols, fatty acids, and resin acids.These extracted compounds can also be inhibitory to metabolic processes,including fermentation, in yeast and other microbes, such as bacteria.

Furthermore, metal cations including calcium, aluminum, potassium, andsodium are found in hemicellulose hydrolysate and heavy metals may bepresent from degradation of the metal vessels due to hydrolysis. Thepresence of such metal cations may also be inhibitory above certainconcentrations.

As made clear from the foregoing discussion, the environment experiencedby microbes in biomass hydrolysate is in stark contrast to a defined,artificial medium where all or most of these additional inhibitors arenot present or are added experimentally one at a time to study theireffects. Indeed, in a biomass hydrolysate the various inhibitorycompounds discussed above, as well as others, may work synergisticallywith one another so that a non-inhibitory amount of a certain compoundmay become inhibitory in the presence of one or more additionalcompounds that are also below their respective individual inhibitoryconcentrations.

Because many secondary products can degrade the fermentation process astheir concentrations increase, prior methods for conversion of biomassinto ethanol have employed a costly conditioning step to remove orreduce the concentration of inhibitors from the hydrolysate prior tofermentation. Furfural, HMF, and acetic acid, as well as phenolics arethe most commonly found inhibitors in biomass hydrolysate. Levels in therange of 0.2-5.0 g/L furfural, 0.2-6.0 g/L HMF, and 3.0-10.9 g/L aceticacid are considered common and may greatly reduce fermentation orprevent it all together. Likewise, concentrations of phenolics in therange of 0.1-10 g/L are common and may be inhibitory. A method commonlyused to ameliorate the toxicity of hydrolysates by reducing HMF andfurfural concentration is pH adjustment through “overliming” withcalcium hydroxide. Overliming is the process whereby lime is addedbeyond that necessary for pH adjustment. Even after overliming, however,high levels of inhibitors may still exist. In addition, overlimingprecludes recovery of secondary products that have high value from thehydrolysate.

In order to deal with the potential for high levels of inhibitorysecondary products often found in biomass hydrolysate—for example,levels that would inhibit the fermentation microbes in their freestate—during the fermentation step 104, the present patent documentteaches processes to protect the fermentation microbes from thedegradation effects of the inhibitors by immobilizing the microbes, andmore preferably immobilizing the microbes in calcium alginate.Immobilization of microbes is the attachment or inclusion in a distinctsolid phase, such as calcium alginate, that permits exchange ofsubstrates, products, inhibitors, etc. with the microbe, but at the sametime separates the microbes from the bulk biomass hydrolysateenvironment. Therefore, the microenvironment surrounding the immobilizedmicrobes is not necessarily the same as that which would be experiencedby their free-cell counterparts. As a result, for example, the presentpatent document teaches processes for immobilizing Pachysolentannophilus and for fermenting pentoses and hexoses in the presence ofinhibitors found in hemicellulose hydrolysate, even at concentrationsthat would inhibit the fermentative microbe in its free state.

By immobilizing the fermentative microbe(s) during the fermentation step104, the need for conditioning the biomass hydrolysate to reduce theconcentration of, or possibly even completely remove, inhibitorysecondary products is significantly ameliorated. This is because theneed to lower the concentration of inhibitory secondary products to thelevels necessary for fermentation using free microbes is eliminated.Thus, as reflected in FIG. 1, conditioning to reduce the concentrationof inhibitors may be omitted in some embodiments, or, as shown in FIG.2, included as an optional conditioning step 110.

Conditioning the biomass hydrolysate in conditioning step 110 to reducethe concentration of inhibitory secondary products may still bedesirable where, for example, the concentration of the secondaryproducts (either individually or in combination) is sufficiently high tointerfere with the fermentation of sugars even by the immobilizedmicrobe(s). In such cases, however, the concentration of the inhibitorysecondary products will generally not need to be reduced to the samelevels as necessary for fermentation using free microbes and thus a lesssevere and less costly conditioning process may be employed. To offsetthe costs associated with the overall fermentation process, it may alsobe desirable to recover secondary products having a high value throughan optional high value secondary product recovery step 114 shown in FIG.2. Following partial removal (and possible recovery) of many secondaryproducts from the biomass hydrolysate, however, the concentrations ofthese products may remain sufficiently elevated within the hydrolysate,particularly considering the synergistic nature of the inhibitors, tointerfere with fermentation of sugars to ethanol or other biofuel by thefermentative microbe(s) in their free state. Accordingly, the use ofimmobilized fermentative microbe(s) in fermentation step 104 is animportant aspect of the processes described herein, even when theoptional conditioning step 110 is employed to reduce the concentrationof secondary products contained in the biomass hydrolysate.

In some instances, it may also be desirable to perform conditioning step110 even when the concentration of inhibitory secondary products isinsufficient to inhibit fermentation by the immobilized microbe(s)where, for example, the secondary products have high value and thus itis desirable to separately recover the high value secondary productsthrough high value secondary product recovery step 114. This may bedesirable, for example, where the net value of the recovered high valuesecondary products may be used to offset, and hence lower, the costsassociated with the overall fermentation process.

There are numerous methods of performing the conditioning step 110 toreduce the concentrations of inhibitory secondary products. Employingdifferent conditioning methods for conditioning step 110 will result indifferent concentration levels of inhibitory secondary productsremaining in the hydrolysate. The method of conditioning chosen forconditioning step 110 may depend on a variety of factors, including thesensitivity of the microbe used during fermentation to inhibitorysecondary products, costs, and whether there is a desire to recover highvalue secondary products during a recovery step 114. The more sensitivethe microbe, the more desirable it will be to reduce the concentrationof the inhibitory products from the biomass hydrolysate duringconditioning of the hydrolysate in step 110. Immobilization of thefermentative microbe(s), however, will decrease the sensitivity of themicrobe to inhibitory secondary products and thus may reduce thecomplexity and costs incurred during conditioning step 110. Some of theconditioning methods that may be employed in conditioning step 110 toreduce the concentration of secondary products include, but are notlimited to: 1) overliming of hydrolysate; 2) activated carbon (AC)treatment followed by pH adjustment; 3) ion exchange followed byoverliming; 4) AC treatment followed by ion exchange; and 5) ACtreatment followed by nanofiltration.

When hydrolysate from solid-liquid separation step 108 contains one ormore high value secondary products, the secondary products may berecovered in step 114 from the hydrolysate and subsequently used forother purposes. Some of the high-value secondary products that may berecovered in step 114 include, but are not limited to, the mineral acidused in the pretreatment process 100, such as sulfuric acid, acetic acidhydrolyzed from hemicellulose polymers, anti-oxidant molecules (phenolicand polyphenolic compounds) liberated from the partial hydrolysis oflignin during hydrolysis step 102, other organic acids, nutraceutical,cosmeceutical, or pharmaceutical products, and different furans andfuran derivatives, such as 5-hydroxymethylfurfural and furfural. Highvalue secondary product recovery step 114 may be accomplished byadsorption of the secondary products to different matrices, includingactivated carbon, ion exchange resin, ion exchange membrane, organicmolecule “scavenging” resins, polystyrene beads, or any other similartype medium with a high surface area. High value secondary productrecovery step 114 may also be accomplished by separating the secondaryproduct(s) from the soluble hexoses and pentoses through ion exclusionchromatography, pseudo-moving bed chromatography, high performanceliquid chromatography or by filtration methods including micro-, nano-,and ultrafiltration using hollow fiber or membrane technologies. Highvalue secondary product recovery step 114 may include several of theaforementioned processes in series to recover different molecularspecies. Furthermore, the recovery process(es) employed in step 114 maybe tailored to recover specific secondary products according to thenature of the starting biomass. Because many of the recovered secondaryproducts (acetic acid, furans and their derivatives, phenolic andpolyphenolic compounds, levulinic acid, formic acid, and others) areinhibitory to yeast and bacterial fermentation of sugars to ethanol,recovery of high value secondary products in step 114 may both increasethe economics of the entire process and allow for more efficientfermentation in step 104 of the pentoses and hexoses.

In general, microbes may be immobilized for fermentation 104 of biomasshydrolysate in step 104 using a number of different methods. Microbesmay be bound to a matrix material or, more preferably, immobilized byentrapment in the matrix material. For example, microbes may beimmobilized by entrapment using a drop-forming procedure. The resultantbeads may be of different size and possess different pore sizes. Forexample, the beads may range in size from 0.1 mm to 5 mm in diameter,more preferably the beads may range from 2 mm to 3 mm in diameter, andmore preferably the beads are about 3 mm in diameter.

The drop-forming procedure may be enhanced through a number ofprocesses. The beads, may be hardened to different degrees and may havecoatings applied to withstand shear forces in a reactor and to reducecell loss. For example, if calcium alginate is used, the beads may bedried to increase compression stress. The beads may also be hardened byglutaraldehyde treatment or coated with catalyst-free polymer to enhancetheir stability. The beads may be recoated with plain alginate as adouble layer to enhance their gel stability. Furthermore, the beads mayhave a polyacrylamide coating to enhance their structural stability. Thebeads may also be coated with a copolymer acrylic resin to increasediffusion and reduce cell leakage. Similarly, other additions to thedrop forming procedure may be incorporated to enhance the effectivenessof the matrix.

Other techniques for improving the efficiency of immobilized microbesinclude increasing the surface area of the microbe/immobilization mediummixture once it is formed. For example, a Pachysolen tannophilus/calciumalginate or other microbe/calcium alginate mixture may be applied as acoating to a natural or synthetic, high surface area, support structure.In one implementation, the support structure only need be able tosupport the microbe/immobilization medium and itself. For example, thesupport structure may comprise a ceramic sponge, honeycomb, reactorpacking material or other support structure to increase the surface areaper mass of the microbe/immobilization medium when it is applied. Themixture may also, or in the alternative, be applied to parts of thereactor surfaces, such as, the walls or the surface of the mixingdevices.

In addition to immobilization by entrapment, the microbes may beimmobilized by other methods including adsorption, cross-linking, orimmobilized by any other means capable of providing a micro-environmentfor the microbe.

A variety of different materials may be used to immobilize microbes. Ifthe microbes are immobilized using entrapment calcium alginate, anatural product from brown algae (seaweed) may be preferably used.However, other materials, both natural and synthetic, may also be usedto immobilize microbes using entrapment including carrageenan, xanthangums, agarose, agar and luffa, cellulose and its derivatives, collagen,gelatin, epoxy resin, photo cross-linkable resins, polyacrylamide,polyester, polystyrene and polyurethane.

Other materials that may be used to immobilize microbes using adsorptionor other immobilization methods include kieselguhr, wood, glass ceramic,plastic materials, polyvinyl acetate, and glass wool.

When combining microbes with complimentary properties, the microbes maybe combined within the same immobilization vehicle, or the microbes maybe immobilized separately and the separately immobilized microbescombined in the same fermentation reactor. For example, if calciumalginate beads are used as the immobilization vehicle, differentcomplimentary microbes may be combined within the same bead. As oneexample, to effectively ferment softwood hydrolysate, which contains thesugars mannose, galactose, glucose and xylose, to ethanol, one maycombine Zymomonas mobilis, NREL strain 8b, which ferments glucose andxylose to ethanol, with Saccharomyces cerevisiae, which ferments mannoseand galactose, into a single bead product. In this way advantageousfermentative properties of different microbial species are combined in asingle bead product.

Alternatively, separate beads can be made containing each microbe andthen the beads may be combined in the fermentation reactor. For example,the fermentation of the hexoses and pentoses to fuel may be performed bycombining beads composed of different microbial species withcomplementary hexose and pentose specificities, metabolic rates, or thelike. In yet another example, different microbes are immobilized inseparate reactors and the biomass hydrolysate is then run through eachreactor to expose the biomass hydrolysate to each microbe. In addition,different immobilization methods may be combined with differentmicrobes.

One of the many advantages of immobilizing the microbes is that themicrobes become more stable and bioreactors may be run in a continuousmode instead of batch mode. Running the bioreactor in a continuous modeis advantageous for efficiency reasons but the microbes may begin tolose metabolic efficiencies after long periods of use. In order torestore metabolic efficiency, immobilized microbes may be periodicallytreated with yeast growth medium. For example, Pachysolen tannophilusand other fermentative microbes immobilized in calcium alginate may beperiodically treated with a yeast growth medium to restore metabolicefficiency.

Another advantage of microbe immobilization is that the microbe biomassmay be better retained within a continuous fermentation reactor. In acontinuous fermentation process involving a high flow rate, such as thatwhich may be experienced during the continuous running of a columnarup-flow reactor, free cells will tend to wash out. Wash out reduces thenumber of cells in the reactor and thus lowers the rate of thefermentation reaction. To maintain the rate of fermentation, new cellsmust be propagated and added to the reactor, increasing costs. Theexamples associated with Table 2 below demonstrate the advantages ofusing immobilized microbes in a continuous fermentation process underwash out conditions (i.e., under a flow rate that would cause wash outof more than 5% of the free cells.)

TABLE 2 Effect of cell washout on ethanol concentration and productivityin a continuous reactor. Retention Ethanol Productivity time (h) Cells(g/L) (g/L · h) 10 Imm 3.03 0.30 Free 1.84 0.18 5 Imm 2.08 0.42 Free0.68 0.14 Imm—immobilized

The data in Table 2 illustrates the benefits of immobilization toprevent wash out for one particular fermentative microbe. Specifically,the example in Table 2 demonstrates the improvement of biofuel (e.g.,ethanol) yield for immobilized Pachysolen tannophilus (NRRL Y2460) overfree cells of the same microbe during continuous fermentation in acolumn up-flow reactor. However, immobilization can be used to preventwash out for any type of fermentative microbe in any continuous flowbioreactor and thereby increase ethanol or other biofuel yield.

The data presented in Table 2 was generated by adding 8.38×10¹¹ cells ofPachysolen tannophilus to two identical up-flow reactors. In the firstreactor, the cells were immobilized in 2-3 mm calcium alginate beads. Inthe second reactor, the cells were added free in solution. Bothreactors, were connected to the same reservoir of artificial medium andthe same peristaltic pump was used to pump the artificial medium throughthe reactors during the continuous fermentation process. The artificialmedium within the reservoir contained 10 g/L yeast extract, 20 g/Lpeptone, 7.2 g/L glucose, and 42.5 g/L xylose. The artificial medium waspumped into the bottom of both reactors simultaneously at the same rateand both reactors were incubated at 30° C.

In a first test, the two reactors were each run at a flow ratecorresponding to a retention time of 10 hours. The reactors were eachrun for a total of 20 hours or for a total of 2× the retention time. Ina second test, set up as indicated above, the two reactors were each runat a flow rate corresponding to a retention time of 5 hours. In thesecond test, the reactors were run for a total of 10 hours, or again fora total of 2× the retention time. The ethanol content of the first andsecond reactor's effluent was analyzed for ethanol content at the end ofthe 2× retention time period for each test. Hence, ethanol content ofeffluent was determined for each reactor at two separate flowconditions. The productivity (ethanol production per hour) was alsodetermined for each flow condition. The results are reported in Table 2.

Table 2 reveals that the ethanol concentration at the end of 20 hoursfor the 10 hour retention time flow rate was much greater for thereactors containing immobilized Pachysolen than free Pachysolen, 3.03versus 1.84 g/L, respectively. The corresponding productivity was alsogreater for the immobilized Pachysolen. For the second test, whichemployed a flow rate that resulted in a 5 hour retention time, theethanol concentration in the effluent of the reactor containingimmobilized cells was 2.08 g/L at the end of 10 hours or 2× theretention time, but the productivity increased by 40% over that in thefirst test due to the faster flow rate.

In contrast, at the flow rate that resulted in a 5 hour retention time,the ethanol concentration in the reactor containing free cells decreasedfrom 1.84 to 0.68 g/L and the productivity experienced a 23% decrease,from 0.18 to 0.14 g/L*hour.

The examples of Table 2 illustrate that immobilizing fermentativemicrobes decreases wash out and increases biofuel, such as ethanol,productivity in the reactor. When the cells were not immobilized, theflow rate of the medium exceeded the sedimentation rate of the freePachysolen tannophilus (at both flow rates tested) and the concentrationof the cells in the free state reactor decreased to a low level causingthe ethanol concentration and ethanol productivity to also decrease. Bycontrast, the Pachysolen tannophilus that was immobilized in the calciumalginate beads remained in the reactor and the reactor was able toincrease the ethanol productivity with the increased flow rate.

Certain microbes that can be used in conversion of sugars to biofuelsare motile; that is, they possess cilia and/or flagella and swim infermentation medium. Another advantage of immobilization is that themotile microbe biomass may be better retained within a continuousfermentation reactor, even in fermentation process involving a low flowrate. Motile cells in the free state will tend to wash out in all flowconditions. Wash out reduces the number of cells in the reactor and thuslowers the rate of the fermentation reaction. To maintain the rate offermentation, new cells must be propagated and added to the reactor,increasing costs.

Another advantage of immobilizing microbes is the ability to obtain ahigh biomass concentration in a continuous fermentation process. In acolumn upflow reactor, as a non-limiting example, more than half,preferably about two thirds to about three quarters of the reactorvolume will be composed of the bead material and the rest will be interparticle void volume when the fermentative microbes are immobilized inbeads of about 2 mm to 3 mm in diameter. In the case of using yeast asthe fermenting microbe, where 5% of the volume of the bead is yeastbiomass, the reactor will effectively contains about 3.3 to 3.75% byvolume yeast biomass, which is a relatively high yeast concentration fora fermentor.

Other benefits of yeast and bacteria immobilization by entrapment incalcium alginate over free cells in suspension include greater ethanoltolerance, possibly due to changes in cell membrane composition; greaterspecific ethanol production, increased rate of ethanol production due toincreased glucose uptake and lower dissolved CO₂ in solution, andincreased thermo-stability of bacteria.

As described above, there are numerous methods of actually immobilizingthe microbes. In one preferred embodiment for immobilizing Pachysolentannophilus in calcium alginate, the microbes are initially immobilizedin sodium alginate which is then converted to calcium alginate. Sodiumalginate can have different viscosities when a given amount is dissolvedin an aqueous solution. Viscosities for different sodium alginateproducts range from 100 or 200 mPa, to even as much as 1236 mPa. In apreferred embodiment, alginate with medium-low viscosity of about 324mPa is used to produce beads, although alginates with differentviscosities may be used for different biomass hydrolysates or forsolid-state ferments.

The sodium alginate is prepared by adding from 0.05 to 10%, orpreferably about 3.5% (w/v) sodium alginate to deionized water.Alternatively, the sodium alginate can be dissolved into growth medium,into a mixture of vitamins, including biotin, or into growth mediumsupplemented with vitamins, or into a natural solution containingbiotin. The initial sodium alginate concentration will depend on thefinal concentration desired to produce beads and on the volume added bymixing with a concentrated microbe slurry.

In order to get some sodium alginate preparations into solution, themixture may be heated and stirred on a stir plate. However, heatingalginate polymers may cause some amount of hydrolysis of the alginateand thereby change the properties of the alginate solution, includingits viscosity. As a result, it may be desirable to use a sodium alginatepreparation that does not require heating in order to go into solution.In embodiments where the alginate may not be heated for solubilizationnor autoclaved for sterilization, it may be desirable to treat thealginate with a chemical sterilizer or it may be desirable to irradiatethe alginate with ultraviolet light for sterilization.

Cells may be cultivated in their respective media, and pelleted bycentrifugation. Alternatively, a mass of Pachysolen or other infermentative microbe may be propagated in at least a 10 L, or morepreferably at least a 200 L, or even more preferably at least a 2000 Lbioreactor to a concentration of about 1 to about 20 grams wet mass perliter growth medium. The resulting biomass may then be concentratedusing, for example, a tangential flow filtration device to produce a20-70% wet mass slurry of Pachysolen cells. This technique isparticularly well suited for the production of large volumes of calciumalginate beads having one or fermentative microbes, such as Pachysolen,immobilized therein.

Following concentration, the concentrated cells are then recovered andthoroughly mixed with the sodium alginate medium. Mixing the alginatewith the microbial cells can occur in the same device as is used for theresuspension of the alginate or in a separate device. The mixingcontinues to homogenity of the mixture. Mixing of the microbes with thehighly viscous sodium alginate solution requires a mixing method thatdoes not shear the microbes, such as a reciprocating disc mixer. Thecell loading into the sodium alginate medium is both organism andsubstrate dependent. For example, a suitable target loading forPachysolen tannophilus in hydrolysate is at least 5 g cells/100 mLsodium alginate medium.

Calcium alginate beads are produced by extruding the sodium alginatemedium/cells into a sterile calcium chloride solution. A peristalticpump and sterilized Master-flex Bulk-Packed Silicone Tubing that has anattached sterile 18 G needle may be used in the extruding process. Theentire process is preferably done aseptically. In an alternativeembodiment that is more suitable where large amounts of immobilizedmicrobe beads are desired to be produced, a sterile 96 hollow 19 gaugepin device may be used in place of an 18 gauge needle. The beads maythen be produced by extrusion and gravity dropping. Other methods mayinclude a so-called Jet Cutter to produce beads from a continuous streamof an alginate/microbe slurry. Other modifications of producing beadsfrom a continuous stream include using electrostatic attraction toproduce droplets, using vibration to produce droplets, using air toproduce droplets, and using a rotating disk atomizer, to name a few.

In order to exchange sodium ions with calcium ions to effectpolymerization of the alginate, beads are dropped in a solutioncontaining calcium chloride. In one method, a 0.22M solution of calciumchloride dihydrate is also prepared in deionized water to receive sodiumalginate/microbe mixture. The sodium alginate medium and calciumchloride solution may both be autoclaved for sterilization purposes. Thebeads may be kept at 4° C. in the calcium chloride solution for about 60minutes to harden. Once the beads have hardened, they are preferablyrinsed several times with sterile deionized water. In a preferredembodiment, the beads are dropped into sterile growth medium containing0.1 to 0.25 M calcium chloride. The growth medium may also containdifferent vitamins or biotin. After about 30 minutes of hardening, thebeads may be either used immediately in a fermentation or may be storedat 4° C. until use. There is no need to rinse beads prior to use orprior to storage when hardening is carried out in such a growth medium.

In certain implementations, it may also be desirable to recyclecomponents of the immobilization processes. The solid calcium alginateused to immobilize microbes in beads or on a support structure maydelaminate, break-up, shear, or otherwise physically degrade afterprolonged use. In addition, the microbe/calcium alginate mixture mayalso become degraded and discolored through repeated use due to thetrapping of contaminants such as extractives, microbial inhibitors, andother materials. Degradation of the structure, whether due to physicaland/or chemical degradation affects the performance of the fermentationprocess. To overcome deleterious effects of this degradation, new orfresh microbe/calcium alginate mixture may be used in the bioreactor toimprove the reactors performance. However, the frequent replacement ofthe mixture may be uneconomical both in terms of the material costsassociated with production of the calcium alginate, but also due to thecost of the lost microbes.

FIG. 3 illustrates a process 140 for recycling calcium alginate used inthe microbe immobilization process. For example, in the case ofPachysolen tannophilus immobilized in calcium alginate beads, thecalcium alginate from the beads used to immobilize the microbes may berecovered and recycled using process 140. In process 140, the degradedmicrobe/calcium alginate mixture 148, is dissociated with a calciumchelator complexed with a monovalent ion 150, such as sodium citrate orpotassium citrate. Step 150 of process 140 dissociates the alginate andliberates the microbes (bacteria or yeast cells). In one preferredembodiment of process 140, step 150 is accomplished by stirring themicrobe/calcium alginate mixture in 20 g/L sodium citrate or potassiumcitrate with a pH 8.2. at room temperature for 15 minutes.

Once the microbes have been liberated and the alginate dissociated, thesolution is filtered to remove the large particulate and microbes(bacteria or yeast cells) in step 152. The filtered solution is thendialyzed, step 154, against a sodium salt 156, such as sodium chloride,to remove the calcium citrate, extractives, and soluble microbialinhibitors 158. The resulting dialysis of the filtered solution with aninorganic salt, such as sodium chloride, regenerates sodium alginate.The toxic materials are removed as waste stream 160. The sodium alginateis concentrated during dialysis and then used again to produce calciumalginate in steps 142, 144, and 146 as described above. In one preferredembodiment, the sodium alginate is used to immobilize Pachysolentannophilus in calcium alginate beads as taught in the above process.

In addition to the use in processes specifically designed to produce analcohol, such as ethanol, the processes of the present patent documentmay be used in conjunction with other processes. For example, thepaper-pulping process usually burns or discards the hemicelluloseportion of the biomass. Using the processes taught herein, however, thehemicellulose may be separated and removed from the biomass andprocessed into ethanol, or other biofuel. Accordingly, the processes ofthe present patent document provide an efficient, cost-effective meansfor converting hemicellulose into ethanol, or other biofuels, in thepaper-pulping, and other, industries. As a further non-limiting example,the processes disclosed in the present patent document may also be usedto ferment monosaccharides, both hexose and pentose, obtained from thesaccharification of sugarcane bagasse.

The following discussion will now be directed to bioreactors designedfor use with immobilized microbes and in particular with immobilizedPachysolen tannophilus.

Fermentation may occur using a number of methods. Preferably the biomasshydrolysate is removed and fermented ex-situ. A variety of bioreactordesigns, including a traditional non-stirred fermenter or stirredfermenter, may be used for the fermentation of the biomass hydrolysateusing immobilized microbes. The reactor may be a submerged reactor orother type of liquid reactor. In order to provide the highest yield, asubmerged reactor is preferable to ferment five-carbon sugars.

In the case of microbes that are immobilized, a packed bed reactor couldbe utilized, or a tankage system similar to that employed forcarbon-in-pulp processes in the gold mining industry could be used. Inthe latter, beads would be moved counter-current to the solution flowand could be easily recovered for regeneration. Thin film reactors mayalso work well with immobilized microbes.

In addition, solid/liquid contactors may be used with immobilizedmicrobes. These types of reactors include ion exchange columns, packedbed reactors, trickle flow reactors, and rotating contactors. Otherreactors that may be used are fluidized-bed and upflow type reactors.

If the entrapment method of immobilization is used, the microbes may beincorporated into a bioreactor using a number of different methods. Inaddition to beads, the matrix/microbe gel may be applied to a supportstructures to increase the effective surface area. These configurationsmay include coating paddle structures, used in stirred tank reactors,rotating contactors, and thin film reactors. The microbes could also beincorporated in large three-dimensional open-cell supports for use intrickle flow reactors or fluidized-bed and upflow reactors.

FIG. 4 illustrates a view of one embodiment of a bioreactor forperforming submerged fermentation of biomass hydrolysate usingimmobilized microbes. Bioreactor 200, which may be referred to as arotating disk contactor, comprises vessel 202, input 204, rotating stirstick 206, outputs 208 and 210, stators 212, and rotors 214.

Although vessel 202 is shown in a vertical configuration it may also behorizontal or in some other orientation. Vessel 202, preferably includesa large opening. For example, vessel 202 may be made of two separablehalves in order to facilitate maintenance access to the stators 212 orrotors 214 located within vessel 202.

In a preferred embodiment, microbes immobilized in a matrix substance,such as calcium alginate, are applied to the stators 212 and the rotors214. With this structure biomass hydrolysate flows through the vessel202 from input 204 and through outputs 208 and 210. While the biomass isflowing, the rotating stir stick 206 may be rotated to provide agitationto the biomass hydrolysate as it flows through the bioreactor 200.Preferably the bioreactor 200 is designed for continuous flowfermentation.

FIG. 5A and FIG. 5B illustrates a side and front view of anotherembodiment of a bioreactor for performing submerged fermentation ofbiomass hydrolysate using immobilized microbes. Bioreactor 300,comprises motor 302, rotating shaft 304, media disk panels 306, biomasshydrolysate 308, vessel 310, and optional air tube 312. Biomasshydrolysate 308, is added to the bioreactor 300 for fermentation.

Vessel 302 of bioreactor 300 is shown as only a bottom half, but vessel302 may completely encapsulate the rotating media disks 306. In apreferred embodiment, microbes immobilized in a matrix substance may beapplied to the media disk panels 306. Motor 302 rotates the media disks306 through the biomass hydrolysate 308.

In one embodiment, bioreactor 300 includes an optional air tube 312 thatmay be used to further agitate the biomass hydrolysate 308 and increasefermentation by injecting air below the rotating media disk panels 306.

FIG. 6 illustrates an upflow reactor. Upflow reactor 400 contains sludgebed or sludge blanket 402. For use to ferment biomass hydrolysate,sludge bed 402 comprises immobilized microbes. Sludge bed 402 may becomprised of one or more fermented microbes immobilized in any of thevarious medium described above. For example, sludge bed 402 may becomprised of Pachysolen tannophilus immobilized in calcium alginatebeads. Upflow reactor 402 further comprises inlet(s) 404 for influent.Inlet(s) 404 may be a single inlet or more preferably a plurality ofinlets across the bottom of the upflow reactor 400 to distribute theinfluent evenly underneath the sludge bed 402. Inlet(s) 404 allow thebiomass hydrolysate to enter the upflow reactor from beneath the sludgebed 402. As the biomass hydrolysate is fermented, biogas 406 rises tothe surface of the reactor and is collected at the top 408 of the upflowreactor 400. Effluent 410 is removed from the reactor and recycledthrough the inlet(s) 404.

Preferably the upflow reactor 400 is a columnar upflow reactor with alow aspect ratio between the range of about 1:1 to 2:1 height to width.Carbon dioxide gas produced by the fermentation process disrupts thepacking of the beads loaded in the column and promotes a‘self-fluidizing’ bed, similar to the effect achieved by a gas-lift typeof reactor.

In a preferred embodiment, two or more ‘self-fluidizing’ bed columnarupflow reactors 400 can be run in series. The beads in each reactors maycontain the same or different microbes, so as to ferment differentsugars in different reactor stages. An increase in the number ofreactors placed in series will reduce the sugar/ethanol variation withinany given reactor, which in turn will promote better microbeperformance.

In addition to the bioreactor designs shown in FIG. 4, FIG. 5A, 5B, andFIG. 6, it is to be understood that numerous other submerged or contactbioreactor designs may be used with the processes taught herein.

Bioreactors based on immobilized microbes offer several advantages over‘free cell’ systems. One advantage is the increased feasibility toemploy a continuous fermentation system. Immobilization ensures no lossof cell mass, such as occurs with batch fermentation and with continuousfermentation where the flow rate is such that the free cells are washedout of the reactor with the product. Continuous fermentation alsodecreases production down-time compared to batch fermentation.Continuous fermentation using microbes immobilized in beads increasesthe flow rate and the ethanol productivity possible with, for example,an upflow reactor. Immobilization also ensures no loss of cell mass ofmotile cells, where the flow rate is either high or low, where theinherent motility of the cell leads to loss of cell mass.

The following example demonstrates the application of one embodiment ofthe present patent document applied to beetle-killed pine. For thepurposes of the present example Pachysolen tannophilus was eitherimmobilized in calcium alginate beads with about a 3 mm diameter(generated using the method describe above) or was in a free cell state.Tables 3 and 4 below summarize the improvement of ethanol yield, and inglucose and xylose conversion resulting from the reactor design employedaccording to the present example.

The present example demonstrates the improvement of ethanol yield, andin glucose and xylose conversion, for calcium alginate-immobilizedPachysolen tannophilus in two different softwood hydrolysates (‘A’ and‘B’) over free (i.e. unrestricted) Pachysolen tannophilus. Thehydrolysates were pH adjusted or overlimed and pH adjusted. ThePachysolen tannophilus strain NRRL Y2460 was used in carrying out theexperiment; however, other adapted or mutated strains of Pachysolentannophilus may also be immobilized in calcium alginate and used inprocesses according to the present patent document.

The pine was transformed into a softwood hydrolysate by dilute acidhydrolysis. The hydrolysate was either simply pH adjusted with sodiumhydroxide or ‘overlimed’. As mentioned above overliming with calciumhydroxide is commonly used to ameliorate the toxicity of hydrolysates.The resulting solutions were fermented using Pachysolen tannophilusimmobilized in 3 mm calcium alginate beads.

The beads were incubated in a flask of Yeast Peptone Dextrose (YPD)broth for 22 hours at 30° C. and 75 rpm. YPD is a standard yeast mediumcontaining 10 g/L yeast extract, 20 g/L peptone, and 20 g/L dextrose.Similarly, the free cells were cultured from a working slant into aflask of YPD broth and incubated for 24 hours at 30° C. and 75 rpm.

To prepare the pH adjusted hydrolysate, the solution was adjusted to pH6.0 with 8M potassium hydroxide, followed by filter sterilization.Preparation of overlimed and pH adjusted hydrolysate required overlimingto pH 10.0 with calcium oxide, followed by a 30 minute hold at 50° C.under stirring conditions. The overlimed hydrolysate was then filteredto remove the solids. Following re-acidification to pH 6.0, thehydrolysate was filter sterilized.

Serum vials were aseptically prepared to obtain a final concentration of95% hydrolysate with the following nutrient additions: 0.2% urea w/v,0.2% yeast extract, and 0.05% potassium dihydrogen phosphate. Theinoculation rate for immobilized beads was 0.2 g beads per mL. Followingrinsing and re-suspension in sterile buffer, the free cells wereinoculated at a rate of 0.3 OD_(600 nm) per mL. All experimentalconditions were set up in triplicate serum vials. The vials wereaseptically vented and incubated for 72 hours at 30° C. and 75 rpm priorto sampling for analysis.

In pH adjusted hydrolysate “A”, as shown in Table 3, ‘free’ Pachysolenwas unable to convert sugars to ethanol and no xylose was utilized.Immobilized Pachysolen converted most of the sugars (81%) to ethanol andconverted 51% of the xylose. The data shows that immobilization greatlyincreased the ability of Pachysolen to overcome the inhibitory effectsof the toxic compounds contained in the pH adjusted hydrolysate.

In overlimed hydrolysate “A”, as reflected in Table 3, ‘free’ Pachysolenconverted 60% of sugars to ethanol, and immobilized Pachysolen 86% ofsugars. Xylose utilization was 0% for free cells. This is a surprisingresult with respect to reports in the current literature that Pachysolentannophilus will ferment pentoses, and particularly xylose, in a definedmedium. It is the inventors' hypothesis that despite removal ofdetectable levels of HMF and furfural by overliming, significant amountsof other inhibitors, discussed above, or combinations thereof stillremain in the hydrolysate thus preventing fermentation. When thePachysolen tannophilus was immobilized xylose utilization jumped to 76%.Immobilization thus enhances the benefit of overliming and greatlyincreases xylose utilization.

Table 4 shows similar results to Table 3. In pH adjusted hydrolysate“B”, as shown in Table 4, ‘free’ Pachysolen was unable to convert sugarsto ethanol and no xylose was utilized. Immobilized Pachysolen converteda majority of the sugars (57%) to ethanol.

Moreover, as reflected in Table 4, in overlimed hydrolysate “B” thatcontained very high inhibitor concentrations, ‘free’ Pachysolen wasunable to ferment available sugars, while immobilized Pachysolenfermented 83% of available sugars, including xylose, to ethanol.

TABLE 3 Softwood hydrolysate ‘A’ fermentation characteristics withPachysolen tannophilus pH adjusted Overlimed and pH adjusted Ethanolyield Sugar Utilization Solution Ethanol yield Sugar UtilizationSolution (% (%) Inhibitors (g/L) (% (%) Inhibitors (g/L) Cellstheoretical)† glucose xylose furfural HMF theoretical)† glucose xylosefurfural HMF Free 0.0% 0.0% 0.0% 0.42 4.03 61.8% 72.2% 0.0% <DL <DL57.4% 75.8% 0.0% Imm. 81.3% 65.2% 51.1% 0.42 4.03 79.7% 61.6% 79.3% <DL<DL 92.7% 67.9% 73.4% †Glucose concentration: 13.5 g/L; Xyloseconcentration: 3.4 g/L DL = Detectable Limit; Imm. = Immobilized

TABLE 4 Softwood hydrolysate ‘B’ fermentation characteristics withPachysolen tannophilus pH adjusted Overlimed and pH adjusted Ethanolyield Sugar Utilization Solution Ethanol yield Sugar UtilizationSolution (% (%) Inhibitors (g/L) (% (%) Inhibitors (g/L) Cellstheoretical)† glucose xylose furfural HMF theoretical)† glucose xylosefurfural HMF Free 0.0% 0.0% 0.0% 5.91 1.32 0.0% 0.0% 0.0% 1.04 0.79 Imm56.9% 35.7% 15.9% 5.91 1.32 83.3% 53.8% 73.0% 1.04 0.79 †Glucoseconcentration: 4.7 g/L; Xylose concentration: 3.2 g/L Imm. = Immobilized

In the preceding example summarized in Tables 3 and 4, and thesubsequent examples in Tables 5-7 below, ethanol yield (% theoretical)is based on glucose and xylose only and is calculated from total glucoseand xylose concentrations before treatment. Other monosaccharides arenot considered. All sugar utilization data is calculated using YSIresults for glucose and xylose. Sugar utilization calculations do notdifferentiate between end products (i.e., includes ethanol, xylitol,biomass) and is calculated as follows (accounting for lost sugars aftertreatment like overliming, autoclaving, etc.):

For Hydrolysate Calculation:

${\% \mspace{14mu} {Sugar} \times {Conversion}} = {\frac{{NS} - {RS}}{TS}*95}$

-   -   NS=Sugar X Concentration after Treatment (i.e., Negative        Control)    -   RS=Residual Sugar X Concentration after Fermentation    -   TS=Total Sugar X Concentration before Treatment

Other embodiments of the processes taught in the present patent documentwill include using different microbes and different conditioningmethods. For example, Tables 5 and 6 illustrate the improvement inethanol yield, and in glucose and xylose conversion, for calciumalginate-immobilized Zymomonas mobilis NREL strain 8b, Pachysolentannophilus (NRRL Y2460), and Pichia stipitis (NRRL Y7124) in sugarcanehydrolysate over free cells of the same. Similar to the examples intables 3 and 4, pH adjusted hydrolysate was compared against anotherconditioning method for both free and immobilized microbes. In contrastto the examples illustrated in tables 3 and 4, the hydrolysate used forthe examples shown in tables 5-7 used hydrolysate derived from sugarcanebagasse instead of hydrolysate derived from softwood. Tables 5 and 6illustrate the benefit of immobilization on a variety of microbesincluding both yeasts and bacterium.

The effects of the different conditioning steps on the concentrations ofsecondary inhibitory products are shown in Table 7. As shown in Table 7,the hydrolysates were conditioned by pH adjustment or by passing thehydrolysate over activated carbon (AC), strong acid ion exchange (IE)resin and weak base ion exchange resin, a treatment hereafter termedAC/IE.

TABLE 5 Percent conversion of glucose and xylose to ethanol. StrainCells pH adjustment AC/IE Z. mobilis, NREL 8b Imm. 31.4 ± 0.9 71.4 ± 1.0Free 22.1 ± 0.7 32.4 ± 1.4 P. tannophilus Imm. 24.8 ± 0.6 63.6 ± 0.4Free  5.7 ± 0.3 50.0 ± 0.2 P. stipitis Imm. 11.9 ± 0.3 54.4 ± 0.7 Free 4.1 ± 0.1 56.4 ± 2.1 Imm. = Immobilized

TABLE 6 Percent xylose utilized in 6 day fermentation. Strain Cells pHadjustment AC/IE Z. mobilis, NREL 8b Imm. 30.8 ± 0.7 75.1 ± 0.4 Free17.5 ± 0.0 17.6 ± 3.2 P. tannophilus Imm. 23.7 ± 1.7 95.8 ± 0.5 Free11.5 ± 0.7 55.9 ± 4.3 P. stipitis Imm. 16.4 ± 3.1 67.3 ± 1.9 Free N.D.61.7 ± 3.0 N.D.—not detected; Imm.—Immobilized;

TABLE 7 Inhibitor Concentrations in differently conditionedhydrolysates. Acetic Formic 5-HMF Furfural Conditioning acid (g/L) acid(g/L) (g/L) (g/L) pH adjustment 10.7 3.8 1.1 3.5 AC/ion exchange 0.1 0.4N.D. N.D. N.D.—Not Detected.

The examples of Table 5-7 were conducted by transforming sugarcanebagasse into a bagasse hydrolysate by dilute acid hydrolysis. Thehydrolysate was conditioned by either simply pH adjusting with sodiumhydroxide or by treating the hydrolysate with activated carbon and thetwo ion exchange resins mentioned above. Namely, the bagasse hydrolysatewas passed over a column containing activated carbon, over a columncontaining a strong acid cation exchange column, and a weak base anionexchange column. The resulting solutions were further separated intothree separate solutions each to be fermented by three differentmicrobes, Zymomonas mobilis NREL strain 8b, Pachysolen tannophilus (NRRLY2460), and Pichia stipitis (NRRL Y7124) respectively. For each of themicrobe solutions, two separate examples were performed, one with themicrobe immobilized in 2-3 mm calcium alginate beads, and the otherusing free microbes. Consequently, there were four differentfermentations for each microbe resulting in 12 total fermentations. Twofermentations with the microbe immobilized, one with a pH adjustedsolution and one with an AC/IE conditioned solution and twofermentations using free microbes, one with a pH adjusted solution andone with an AC/IE conditioned solution.

The two differently-conditioned bagasse hydrolysates contained differentamounts of the inhibitors acetic acid, formic acid, 5-hydroxyfurfural(5-HMF), and furfural. The measured values are reported in Table 7.These inhibitor levels are for the particular batch of sugarcane bagassehydrolysate used in the experiments summarized above for which theresults are reported in Tables 5 and 6.

The beads used for immobilizing the different microbes were incubated ina flask of Yeast Peptone Dextrose (YPD) broth for 22 hours at 30° C. and75 rpm. Similarly, the free cells were cultured from a working slantinto a flask of YPD broth and incubated for 24 hours at 30° C. and 175rpm.

Serum vials were aseptically prepared to obtain a final concentration of95% hydrolysate with the following nutrient additions: 0.2% urea w/v,0.2% yeast extract, and 0.05% potassium dihydrogen phosphate. Theinoculation rate for beads was 0.2 g beads per mL. Following rinsing andre-suspension in sterile buffer, the free cells were inoculated at arate of 0.01 g (wet weight) per mL for P. tannophilus and P. stipitis,and 0.006 g (wet weight) per mL for Z. mobilis 8b. All experimentalconditions were set up in triplicate serum vials. The vials wereaseptically vented and incubated for 6 days at 30° C. and 75 rpm priorto sampling for analysis.

For sugarcane bagasse hydrolysate conditioned by pH adjustment, ‘free’Zymomonas was able to convert 22% of the glucose and xylose to ethanol,while immobilized Zymomonas converted 31% (Table 5). Similarly, ‘free’Pachysolen was able to convert 6% of the glucose and xylose to ethanol,while immobilized Pachysolen converted 25%, and ‘free’ Pichia was ableto convert 4% of the glucose and xylose to ethanol, while immobilizedPichia converted 12% (Table 5). The data shows that immobilizationgreatly increased the ability of Zymomonas, Pachysolen, and Pichia toovercome the inhibitory effects of the toxic compounds contained in thepH adjusted bagasse hydrolysate (Table 5).

In AC/IE conditioned bagasse hydrolysate, as reflected in Table 5,‘free’ Zymomonas was able to convert 32% of the glucose and xylose toethanol, while immobilized Zymomonas converted 71%. Similarly, ‘free’Pachysolen was able to convert 50% of the glucose and xylose to ethanol,while immobilized Pachysolen converted 64%. Unlike Zymomonas andPachysolen, immobilized Pichia was actually less effective at convertingglucose and xylose to ethanol than ‘free’ Pichia. As shown in Table 5,‘free’ Pichia was able to convert 56% of the glucose and xylose toethanol, while immobilized Pichia converted 54%. The data shows thatimmobilization greatly increased the ability of Zymomonas and Pachysolento overcome the inhibitory effects of the toxic compounds contained inthe AC/IE conditioned bagasse hydrolysate.

Xylose utilization in the fermentations generally mirrored the extent offermentation of glucose and xylose to ethanol. Immobilized Zymomonasutilized 31% of xylose in pH adjusted hydrolysate and 75% in AC/IEconditioned hydrolysate, while the free cells utilized only 18% of thexylose in both conditions (Table 6). Immobilized Pachysolen utilized 24%of xylose in pH adjusted hydrolysate and 96% in AC/IE conditionedhydrolysate, while the free cells utilized only 12% and 56% of thexylose, respectively (Table 6). Immobilized Pachysolen utilized 25% ofxylose in pH adjusted hydrolysate and 64% in AC/IE conditionedhydrolysate, while the free cells utilized only 6% and 50%,respectively. Immobilized Pichia utilized 16% of xylose in pH adjustedhydrolysate and 67% in AC/IE conditioned hydrolysate, while the freecells utilized no xylose in pH adjusted hydrolysate, but 62% in AC/IEconditioned hydrolysate (Table 6).

It is the inventors' hypothesis that despite removal of detectablelevels of HMF and furfural and a great decrease in acetic and formicacids by AC/IE conditioning, significant amounts of other inhibitors,discussed above, and the remaining formic and acetic acids, orcombinations thereof still remain in the hydrolysate thus interferingwith fermentation. For Zymomonas and Pachysolen, immobilizationincreased xylose utilization significantly. Immobilization thus enhancesthe benefits of conditioning and greatly increases xylose utilization.

In another example of the processes taught in the present patentdocument, the microbe/calcium alginate beads were re-used in sequentialfermentations and the microbes in the beads were metabolically‘regenerated’ between fermentations to increase ethanol yield.

For the present example, fermentations using 2 g Pachysolen/calciumalginate beads per 10 ml softwood hydrolysate supplemented with 0.2%Urea, 0.2% Yeast Extract, and 0.05% KH₂PO₄ were performed at 30° C. and75 rpm for 72 hours. After the fermentation reaction (Fermentation 1),the liquid was aseptically removed and analyzed for ethanol content, andthe beads were aseptically rinsed several times with sterile deionizedwater. The same Pachysolen/calcium alginate beads were used in a secondfermentation (Fermentation 2), in the same conditions, asFermentation 1. Similarly, the fermentation liquid was subsequentlyanalyzed and the beads rinsed. This was repeated for Fermentation 3.FIG. 7 illustrates the decreased ethanol yield in Fermentations 2 and 3compared to Fermentation 1.

Next, the same Pachysolen/calcium alginate beads were regeneratedbetween Fermentations 3 and 4 (shown as a dotted line in FIG. 7 betweenFermentations 3 and 4) by incubating for 22 hours in a shaking incubatorat 30° C. and 100 rpm in a yeast culture medium, Yeast Peptone Dextrose(YPD), after washing. The YPD was then aseptically removed and the beadswere used in yet another fermentation (Fermentation 4). FIG. 7illustrates that the regeneration of the Pachysolen/calcium alginate inculture medium restored the fermentative ability of the Pachysolen toproduce ethanol.

Similar washes, fermentations, and a second regeneration (shown as adotted line between fermentations 7 and 8) were performed using the samebeads in another 6 fermentations. The results are shown in FIG. 7. FIG.7 illustrates that immobilized microbes may be used in sequentialfermentations and that the Pachysolen in the beads can be metabolicallyregenerated. Although the present example employs a regeneration stepafter 3 or 4 consecutive uses of the immobilized microbes, it ispossible to regenerate the microbes more or less often. It is expectedthat if a greater number of beads are used in sequential fermentations(i.e. fermenting under conditions of a saturating yeast concentration),the ethanol yields would remain at a higher level in successivefermentations before requiring metabolic regeneration.

As discussed above, the immobilization medium, for example calciumalginate, can degrade due to use. If the microbes are regenerated andre-used according to the present example, it may be necessary to recyclethe immobilization medium as taught above.

Although the invention has been described with reference to preferredembodiments and specific examples, it will readily be appreciated bythose skilled in the art that many modifications and adaptations of themethods and bioreactors described herein are possible without departurefrom the spirit and scope of the invention as claimed hereinafter. Thus,it is to be clearly understood that this description is made only by wayof example and not as a limitation on the scope of the invention asclaimed below.

What is claimed is:
 1. A medium for fermenting biomass hydrolysate, themedium comprising: calcium alginate beads ranging from 0.1 mm to 5 mm indiameter; a microbe strain capable of fermenting pentoses immobilized inthe calcium alginate beads, wherein the immobilized microbe strain iscapable of converting at least 70% of available pentoses in a biomasshydrolysate to a biofuel.
 2. A medium for fermenting biomasshydrolysate, the medium comprising: an immobilization substance capableof providing a micro environment for a microbe strain; a microbe straincapable of fermenting pentoses into a biofuel immobilized in theimmobilization substance, wherein the microbe strain comprises about 5%by volume of the immobilization substance.